IL-12 enhancement of immune responses to T-independent antigens

ABSTRACT

The present invention relates to a method of modulating an immune response to a T-cell or thymus independent antigen in a host (e.g., mammalian, including human), comprising administering to the host an effective amount of interleukin-12 (IL-12) and the T-cell independent antigen. In one embodiment, the present invention relates to a method of inducing an immune response to a TI antigen in a host (e.g., mammalian, including human), which comprises administering to the host an effective amount of interleukin-12 (IL-12) and the TI antigen. In another embodiment, the present invention relates to a method of enhancing an immune response against a TI antigen in a host, which comprises administering to the host an effective amount of IL-12 and the TI antigen. The methods of the present invention can be used, for example, to induce and or enhance a humoral immune response (IgG2a and/or IgG3 humoral immune response).

RELATED APPLICATION(S)

[0001] This application is a continuation of application Ser. No.09/035,594, filed Mar. 5, 1998. The entire teachings of the aboveapplication(s) are incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by grant R21AI38380 from the National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Non-protein antigens such as polysaccharides and lipids induceantibody responses without the need for T cells and are thereforereferred to as T-independent (TI) antigens. However, because of the lackof involvement of T cell help, most TI antigens are relatively poorimmunogens. In general, responses to TI antigens consist of IgMantibodies of low affinity, and do not show significant heavy chainclass switching, affinity maturation, or memory. The practicalsignificance of TI antigens is that many bacterial capsular and cellwall polysaccharides belong to this category and are thereforerelatively poor at eliciting humoral immunity.

[0004] Young children and the elderly are particularly susceptible tolife-threatening infections with encapsulated bacteria such aspneumococcus and meningococcus. It has been estimated by the Centers forDisease Control that in the U.S. per year, Streptococcus pneumoniaecauses 3,000 cases of meningitidis, 50,000 cases of bacteremia, 500,000cases of pneumonia, and 7 million cases of otitis media (middle earinfection). The World Health Organization has estimated that worldwide,this organism causes 100 million cases per year with 10 million deathsper year. Similarly, Neisseria meningiditis is the leading cause ofmeningitis in children and young adults with 2,600 cases/year in theU.S., 310,000 cases and 35,000 deaths per year worldwide.

[0005] Polysaccharide vaccines for inducing immunity to pathogens suchas S. pneumoniae and N. meningiditis are available, but they aregenerally ineffective in children less than 2 years of age and are oflimited efficacy in older individuals. In addition, in all recipientsthe vaccines, even in conjugate form, induce limited isotype switching.Clearly, alternative approaches for vaccination against pathogens havingTI antigens are needed.

SUMMARY OF THE INVENTION

[0006] Applicants have found that interleukin-12 (IL-12) serves as avery strong adjuvant for eliciting immune responses (e.g., antibody(IgG) response) during T-cell independent (TI) immune responses,including responses to vaccine preparations which are currently used inhumans. Thus, the present invention relates to a method of enhancing animmune response against a TI antigen (one or more) in a host. In oneembodiment, the present invention relates to a method of inducing animmune response against a TI antigen in a host, which comprisesadministering to the host an effective amount of IL-12 and the TIantigen. In another embodiment, the present invention is a method ofenhancing an immune response against a TI antigen in a host, whichcomprises administering to the host an effective amount of IL-12 and theTI antigen. The methods of the present invention can be used to induceand/or enhance an immune response to a TI antigen in a mammalian host,such as a primate (e.g., human), murine, feline, canine, bovine orporcine host. The invention also relates to compositions comprisingIL-12 and a TI antigen.

[0007] The methods of the present invention can be used, for example, toinduce and/or enhance a humoral immune response (e.g., IgG2a and/or IgG3humoral immune response) in the host. The TI antigen can include, forexample, a carbohydrate (e.g., a polysaccharide), a lipid, (e.g.,liposomes, phosphorylcholine) a glycoprotein, a hapten-carrierconjugate, a lipopolysaccharide, or a phage (e.g., T4). The IL-12 and/orthe TI antigen can be administered as a protein or as a polynucleotideunder conditions in which the TI antigen and/or IL-12 is expressed invivo.

[0008] In a particular embodiment, the present invention relates to amethod of inducing and/or enhancing an immune response to Streptococcuspneumoniae in a host, which comprises administering to the host aneffective amount of IL-12 and the TI antigen of Streptococcuspneumoniae. In another embodiment, the invention relates to a method ofinducing and/or enhancing an immune response to Neisseria meningiditisin a host, which comprises administering to the host an effective amountof IL-12 and the TI antigen of Neisseria meningiditis.

[0009] The invention also encompasses a composition comprising IL-12 anda TI antigen. One or more TI antigens can be used in the methods andcompositions of the present invention.

[0010] Use of IL-12 as described herein provides effective methods andcompositions which can be used to induce and/or enhance an immuneresponse against a TI antigen.

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIGS. 1A-1B are graphs of reciprocal serum dilution versusoptical density (O.D.) showing increased levels of dinitrophenyl(DNP)-specific IgG2a (FIG. 1A) and IgG3 (FIG. 1B) in BALB/c miceinjected with IL-12 and 50 μg of DNP-ovalbumin (DNP-OVA) (open symbols),a model T cell dependent (TD) antigen, compared to control mice injectedwith DNP-OVA and phosphate buffered saline (PBS) (closed symbols); eachline represents binding of serum from an individual mouse.

[0012] FIGS. 1C-1D are graphs of reciprocal serum dilution versus O.D.showing increased levels of DNP-specific IgG2a (FIG. 1C) and IgG3 (FIG.1D) in BALB/c mice injected with IL-12 and 50 μg of DNP-Ficoll (opensymbols) compared to control mice injected with DNP-Ficoll and PBS(closed symbols); each line represents binding of serum from anindividual mouse.

[0013]FIG. 2A-2F are graphs of reciprocal serum dilution versus O.D.showing the effect of treating BALB/c mice with 20 μg of MenomuneA/C/Y/W-135 and either IL-12 (open symbols) or PBS vehicle (closedsymbols) on total, IgM, IgG2b, IgG1, IgG2a and IgG3 antibody levels;each line represents binding of serum from an individual mouse.

[0014]FIG. 3A-3E are graphs showing the effect of treating BALB/c micewith PNU-Inmmune and either IL-12 (open symbols) or PBS vehicle (closedsymbols) on total, IgM, IgG1, IgG2a and IgG3 antibody levels; eachsymbol represents binding of serum from an individual mouse.

[0015] FIGS. 4A-4B are graphs of reciprocal serum dilution versus O.D.showing levels of total and IgG2a antibody levels in C57BL/6 micetreated with DNP-Ficoll and either IL-12 (open symbols) or PBS (closedsymbols); each line represents binding of serum from an individualmouse.

[0016] FIGS. 4C-4D are graphs of reciprocal serum dilution versus O.D.showing levels of total and IgG2a antibody levels in C57BL/6 T cellreceptor knockout mice specifically lacking T cells (TCR KO mice)treated with DNP-Ficoll and either IL-12 (open symbols) or PBS (closedsymbols); each line represents binding of serum from an individualmouse.

[0017] FIGS. 5A-5B are graphs of reciprocal serum dilution versus O.D.showing levels of IgG2a and IgG3 antibody levels in (C57BL/6×CBA)F₁control mice treated with DNP-Ficoll and either IL-12 (open symbols) orPBS (closed symbols); each line represents binding of serum from anindividual mouse.

[0018] FIGS. 5C-5D are graphs of reciprocal serum dilution versus O.D.showing levels of IgG2a and IgG3 antibody levels in CD3ε mice lacking Tand NK cells treated with DNP-Ficoll and either IL-12 (open symbols) orPBS (closed symbols); each line represents binding of serum from anindividual mouse.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Polysaccharide vaccines to encapsulated bacteria such asNeisseria meningitidis and Streptococcus pneumoniae are weaklyimmunogenic due to their TI nature. Even when converted to T-dependentforms through conjugation to foreign proteins, polysaccharides induceresponses that are deficient in many respects, such as induction ofmurine IgG2a antibody, the isotype which mediates optimal complementfixation and opsonization. As described herein, IL-12 treatment of miceinduced significantly increased levels of IgG2a antibody to a model TIantigen, DNP-Ficoll, and to vaccines composed of polysaccharides frompneumococci and meningococci. Use of immunodeficient mice lacking Tcells and/or NK cells demonstrated that such cells were not responsiblefor the observed antibody enhancement. Furthermore, the use of IFN-γ KOmice showed that stimulation of TI antibody responses by IL-12 was onlypartially dependent on IFN-γ. The ability of IL-12 to dramaticallyenhance TI antibody responses shows that IL-12 is useful as a powerfuladjuvant to induce protective immune responses against encapsulatedpathogens.

[0020] The present invention relates to a method of enhancing an immuneresponse against a TI antigen in a host. In one embodiment, the presentinvention relates to a method of inducing an immune response against aTI antigen (one or more) in a host, which comprises administering to thehost an effective amount of IL-12 and the TI antigen. In anotherembodiment, the present invention is a method of enhancing an immuneresponse against a TI antigen in a host, which comprises administeringto the host an effective amount of IL-12 and the TI antigen.

[0021] As used herein, the terms “enhance” and/or “enhancing” refer tothe strengthening (augmenting) of an existing immune response to apathogen. The term also refers to the initiation of (initiating,inducing) an immune response to a pathogen.

[0022] As used herein a “T-cell independent (TI) antigen”, also referredto herein as a “thymus-independent antigen”, is an antigen which iscapable of inducing an immune response in a host without the need formature T-cells. Therefore, TI antigens include antigens recognized byimmature T cells (e.g., CD1 molecules), such as lipoarabinomannan. TIantigens also include, for example, carbohydrates (e.g.,polysaccharides), lipids, glycolipids, carrier conjugates (e.g., H.influenza conjugate vaccine, polysaccharide conjugate, lipid conjugate,phage conjugate), lipopolysaccharides and phages (see, for example,Bondada and M.Grag, “Thymus-Independent Antigens” in The Handbook of Band T Lymphocytes, E. Charles Snow, Academic Press, Inc., San Diego,(1994) pages 343-370). Particular examples of TI antigens includebacterial polysaccharides, such as bacterial capsular polysaccharides(e.g., Streptococcus pneumoniae capsular polysaccharide, such as thePNU-Immune 23 vaccine, Neisseria meningiditis A, C, Y and W-135serogroups), and bacterial cell wall polysaccharides (e.g.,streptococcal carbohydrates, phosphorylcholine), liposomes,phosphorylcholine and T4.

[0023] The TI antigen can be obtained or derived from a variety ofpathogens or organisms, such as encapsulated organisms (e.g., bacteriasuch as S. pneumoniae, N. meningiditis, Haemophilus influenzae, Brucellaabortis), viruses (e.g., T4 phage), parasites, fungi and yeast, againstwhich an immune response is desired. The TI antigen of a pathogen can beobtained using skills known in the art. For example, the TI antigen canbe isolated (purified, essentially pure) directly from a pathogen,derived using chemical synthesis or obtained using recombinantmethodology. In addition, the TI antigen can be obtained from commercialsources, as described in the exemplification.

[0024] IL-12 is a recently characterized heterodimeric cytokine that hasa molecular weight of 75 kDa and is composed of disulfide-bonded 40 kDaand 35 kDa subunits. It is produced by antigen presenting cells such asmacrophages, and binds to receptors on activated T, B and NK cells(Desai, B. B., et al., J. Immunol., 148:3125-3132 (1992); Vogel, L. A.,et al., Int. Immunol., 8:1955-1962 (1996)). It has several effectsincluding 1) enhanced proliferation of T cells and NK cells, 2)increased cytolytic activities of T cells, NK cells, and macrophages, 3)induction of IFN-γ production and to a lesser extent, TNF α and GM CSF,and 4) activation of TH1 cells (Trinchieri, G., et al., Blood,84:4008-4027 (1994). IL-12 has been shown to be an importantcostimulator of proliferation in Th1 clones (Kennedy et al., Eur. J.Immunol. 24:2271-2278, 1994) and leads to increased production of IgG2aantibodies in serum (Morris, S. C., et al., J. Immunol. 152:1047 (1994);Germann, T. M., et al., Eur. J. Immunol., 25:823-829 (1995); Sher, A.,et al., Ann. N.Y. Acad. Sci., 795:202-207 (1996); Buchanan, J. M., etal., Int. Imm., 7:1519-1528 (1995); Metzger, D. W., et al., Eur. J.Immunol., 27:1958-1965 (1997)). Administration of IL-12 can alsotemporarily decrease production of IgG1 antibodies (Morris, S. C., etal, J. Immunol. 152:1047 (1994); McKnight, A. J., J. Immunol. 152:2172(1994); Buchanan, J. M., et al., Int. Imm., 7:1519-1528 (1995)),indicating suppression of the Th2 response. The purification and cloningof IL-12 are disclosed in PCT publication nos. WO 92/05256 and WO90/05147, and in European patent publication no. 322,827 (identified as“CLMF”).

[0025] As used herein, “interleukin-12” and “IL-12” refer tointerleukin-12 protein, its individual subunits, multimers of itsindividual subunits, functional fragments or portions of IL-12, andfunctional equivalents and/or analogues of “interleukin-12” and “IL-12”.As defined herein, functional fragments of IL-12 are fragments whichmodulate an immune response against a TI antigen in a host. As alsodefined herein, functional equivalents or fragments of “interleukin-12”and “IL-12” include modified IL-12 protein such that the resulting IL-12product has activity similar to the IL-12 described herein (e.g.,inducing and/or enhancing an immune response to a TI antigen).Functional equivalents or fragments of “interleukin-12” also includenucleic acid sequences (e.g., DNA, RNA) and portions thereof, whichencode a protein or peptide having the IL-12 function or activity (e.g.,inducing and/or enhancing an immune response to a TI antigen). Inaddition, the term includes a nucleotide sequence which through thedegeneracy of the genetic code encodes a similar peptide gene product asIL-12 and has the IL-12 activity described herein. For example, afunctional equivalent of “interleukin-12” and “IL-12” includes anucleotide sequence which contains a “silent” codon substitution (e.g.,substitution of one codon encoding an amino acid for another codonencoding the same amino acid) or an amino acid sequence which contains a“silent” amino acid substitution (e.g., substitution of one acidic aminoacid for another acidic amino acid).

[0026] IL-12 suitable for use in the methods and compositions of thepresent invention can be obtained from a variety of sources orsynthesized using skills known in the art. For example, IL-12 can bepurified (isolated, essentially pure) from natural sources (e.g.,mammalian sources, such as murine or human sources), produced bychemical synthesis or produced by recombinant DNA techniques. Inaddition, the IL-12 can be obtained from commercial sources.

[0027] An effective amount of IL-12 is administered in the methods ofthe present invention which is an amount that induces and/or enhances animmune response to a TI antigen in the host. Thus, as used herein, “aneffective amount of IL-12” is an amount such that when administered to ahost, it results in an immune response or an enhanced immune response tothe TI antigen in the host relative to the immune response to the TIantigen in a host when an effective amount of IL-12 is not administeredto a host. That is, an “effective amount” of IL-12 is an amount thatinduces and/or enhances an immune response to a TI antigen relative tothe immune response to the TI antigen if IL-12 is not administered.

[0028] The IL-12 and the TI antigen can be administered as aprophylactic vaccine or a therapeutic vaccine. That is, the IL-12 can beadministered either before (to prevent) or after (to treat) the effectsof a pathogen having a TI antigen which has appeared and/or manifestedin a host. Thus, the IL-12 can be administered to a host who eitherexhibits the disease state caused by a pathogen from which the TIantigen is obtained or derived, or does not yet exhibit the diseasestate caused by a pathogen from which the TI antigen is obtained orderived. Thus, the IL-12 and TI antigen can be administered to hostseither before or after the disease state is manifested in the host andcan result in prevention, amelioration, elimination or a delay in theonset of the disease state caused by the pathogen from which the TIantigen is obtained or derived.

[0029] The IL-12 and the TI antigen can be administered to a host in avariety of ways. The routes of administration include intradermal,transdermal (e.g., slow release polymers), intramuscular,intraperitoneal, intravenous, subcutaneous, oral, epidural andintranasal routes. Any other convenient route of administration can beused, for example, infusion or bolus injection, or absorption throughepithelial or mucocutaneous linings. In addition, the IL-12 and/or TIantigen can be administered together with other components orbiologically active agents, such as adjuvants (e.g., alum),pharmaceutically acceptable surfactants (e.g., glycerides), excipients(e.g., lactose), carriers, diluents, liposomes and vehicles. If desired,certain sweetening, flavoring and/or coloring agents can also be added.

[0030] Further, the IL-12 and/or the TI antigen can be administered byin vivo expression of polynucleotides encoding such into a mammaliansubject. For example, the IL-12 and/or TI antigen can be administered toa host using a live vector, wherein the live vector containing IL-12and/or TI antigen nucleic acid sequences are administered underconditions in which the IL-12 and/or TI antigen are expressed in vivo.For example, a host can be injected with a vector which encodes andexpresses a TI antigen in vivo in combination with IL-12 protein orpeptide, or in combination with a vector which encodes and expressesIL-12 protein in vivo. Alternatively, a host can be injected with avector which encodes and expresses IL-12 in vivo in combination with aTI antigen conjugated to a peptide or protein form or a mimic of aprotein or peptide form, or in combination with a vector which encodesand expresses a TI antigen. A single vector containing the sequencesencoding a TI antigen and the IL-12 protein are also useful in themethods of the present invention.

[0031] Several expression vector systems are available commercially orcan be reproduced according to recombinant DNA and cell culturetechniques. For example, vector systems such as the yeast or vacciniavirus expression systems, or virus vectors can be used in the methodsand compositions of the present invention (Kaufman, R. J., A J. of Meth.in Cell and Molec. Biol., 2:221-236 (1990)). Other techniques usingnaked plasmids or DNA, and cloned genes encapsulated in targetedliposomes or in erythrocyte ghosts, can be used to introduce IL-12and/or TI antigen polynucleotides into the host (Freidman, T., Science,244:1275-1281 (1991); Rabinovich, N. R., et al., Science, 265:1401-1404(1994)). The construction of expression vectors and the transfer ofvectors and nucleic acids into various host cells can be accomplishedusing genetic engineering techniques, as described in manuals likeMolecular Cloning and Current Protocols in Molecular Biology, which arehereby incorporated by reference, or by using commercially availablekits (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Press,1989; Ausubel, F. M., et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Interscience, 1989).

[0032] As described herein, administration of IL-12 and a TI antigenelicits or enhances an immune response in the recipient host. Inparticular, a humoral immune response against the TI antigen is inducedor enhanced in the host. In one embodiment, the humoral immune responseproduced by administration of IL-12 and a TI antigen results in enhancedlevels of total antibody in the recipient host compared to a host whichdoes not receive IL-12 and the TI antigen. In another embodiment, thehumoral immune response produced by administration of IL-12 and a TIantigen results in production of TI-specific antibody in the host. In aparticular embodiment, the TI-specific antibody response producesspecific IgG2a and/or IgG3 antibody in the recipient host. As shown inthe examples, IL-12 is particularly active in enhancing production ofIgG2a, the antibody isotype that is most effective in complementfixation and opsinophagocytosis, the two mechanisms that are mosteffective in bacterial elimination. It is likely that IL-12 induces orenhances other antibody isotypes such as IgA and IgM.

[0033] The immune response to the TI antigen in the host can be due to ageneral enhanced humoral immune response and/or due to a specifichumoral immune response to the TI antigen. In the methods of inducing orenhancing an immune response to a TI antigen in a host, an effectivetherapeutic amount of IL-12 is administered to the host, which is anamount that induces or enhances an immune response to the TI antigen inthe host and results in the improved condition of the host (i.e., thedisease or disorder caused by the presence of the pathogen from whichthe TI antigen is obtained or derived, is prevented, eliminated ordiminished). The amount of IL-12 used to induce or enhance an immuneresponse to a TI antigen in a host will vary depending on a variety offactors, including the size, age, body weight, general health, sex anddiet of the host, and the time of administration, duration or particularqualities of the disease state. Suitable dose ranges of IL-12 aregenerally about 0.5 μg to about 150 μg per kg body weight. In oneembodiment, the dose range is from about 2.75 μg to about 100 μg per kgbody weight. In another embodiment, the dose range is from about 5 μg toabout 50 μg per kg body weight. Effective dosages may be extrapolatedfrom dose-response curves derived in vitro or animal model test systems.

[0034] In the methods of the present invention, an effective amount ofIL-12 is administered in combination with a TI antigen. That is, thelL-12 is administered at a time closely related to immunization of thehost with a TI antigen, so that an immune response to the TI antigen isinduced or enhanced in the host relative to the immunization of a hostin which IL-12 is not administered. Thus, the IL-12 can be administeredprior to, preferably just prior to, immunization; at the time ofimmunization (i.e., simultaneously); or after immunization(subsequently). In addition, the IL-12 can be administered prior toimmunization with the TI antigen followed by subsequent administrationsof IL-12 after immunization with the TI antigen.

[0035] As described herein, IL-12 is capable of dramatically enhancingTI antibody responses in a manner similar to its effects on TDresponses. Using DNP-Ficoll and bacterial polysaccharides as model TIantigens, it was found that IgG2a and IgG3 antibody responses wereparticularly stimulated by IL-12. Surprisingly, enhancement was observedin mice deficient in both T and NK cells. Furthermore, enhancement ofIgG3 antibody expression occurred independently from IFN-γ andenhancement of IgG2a expression was only partially dependent on IFN-γ(see the Table). The results demonstrate that IL-12 is useful forinducing protective responses against bacterial pathogens.

[0036] IL-12 was found to have similar effects on TD and TI responses tothe DNP hapten. In both cases, specific IgG2a and IgG3 anti-DNP serumlevels were significantly increased by simultaneous administration ofantigen and IL-12 while IgG1 expression was not affected at the timepoints analyzed. Use of TCRβ⁻δ⁻ double KO mice confirmed the TI natureof the response to DNP-Ficoll and the fact that the mechanism for IL-12mediated enhancement did not involve T cells. However, the observedeffects of the IL-12 in the responses of both WT and TCR KO mice toDNP-Ficoll was of a lesser magnitude than that observed in WT miceagainst DNP-OVA. This likely reflects a property of the individualDNP-Ficoll preparation rather than the fact that it is a TI antigensince the use of other TI antigens such as bacterial capsularpolysaccharides yielded levels of IL-12 enhancement similar to thoseseen with TD antigens (Germann, T., et al., Eur. J. Immunol., 25:823-829(1995); Buchanan, J. M., et al., Int. Immunol., 7:1519-1528 (1995)). Ithas also been recently demonstrated that SCID mice reconstituted withhuman peripheral blood lymphocytes could mount primary antibodyresponses to N. meningitidis serogroup C polysaccharide if the mice weretreated with human IL-12 at the time of cell transfer (Westerink, M. A.,et al., J. Infect. Dis., 175:84-90 (1997)). However, in thoseexperiments, it was unclear whether IL-12 was actually stimulatingspecific antibody-producing B cells or simply aiding in engraftment ofthe transferred population. It was previously established that IL-12enhances in vivo TD production of IgG2a in response to protein andhapten-carrier antigens (Morris, S. C., et al., J. Immunol.,152:1047-1056 (1994); Germann, T., et al., Eur. J. Immunol., 25:823-829(1995); Buchanan, J. M., et al., Int. Immunol., 7:1519-1528 (1995);Wynn, T. A., et al., J. Immunol., 157:4068-4078 (1996); Bliss, J., etal., J. Immunol., 156:887-894 (1996); Metzger, D. W., et al., Eur. J.Immunol., 27:1958-1965 (1997)). Administration of the cytokine suppressIgG1 production but this suppression is only temporary and IgG1production is also eventually somewhat enhanced (Germann, T., et al.,Eur. J. Immunol., 25:823-829 (1995); Buchanan, J. M., et al., Int.Immunol., 7:1519-1528 (1995)). While TD antigens stimulate conventionalB cells, TI antigens are thought to preferentially activate cells withthe B-1 phenotype (Cong, Y. Z., et al., Int. Immunol., 3:467-476 (1991)and fail to induce isotype switching. Since B-1 cells inhibit responsesby conventional B cells (Riggs, J. E., et al., J. Exp. Med., 172:475-485(1990)) and IL-12 inhibits B-1 cell function (Vogel, L. A., et al., Eur.J Immunol., 26:219-223 (1996); Velupillai, P., et al., Infect. Immun.,64:4557-4560 (1996)), one influence of IL-12 may be in allowingconventional B cells to respond to TI antigens, thus resulting in theobserved enhancement of IgG production.

[0037] Several groups have reported that NK cells play a major role inthe stimulation of IgG TI responses through release of IFN-γ. It hasbeen shown that Ig secretion induced in vivo or in vitro in a TI mannercan be increased by NK cell activation (Wilder, J. A., et al., J.Immunol., 156:146-152 (1996)) and inhibited by NK cell depletion(Snapper, C. M., et al., J. Immunol., 152:4884-4892 (1994); Wilder, J.A., et al., J. Immunol., 156:146-152 (1996)). Antibody neutralization ofIFN-γ reverses the influence of NK cells (Snapper, V. M., et al., J.Immunol., 157:2229-2233 (1996). Recently, a role for endogenous IL-12 inTI responses was proposed by Koh and Yuan (Koh, C. Y. and Yuan, D., J.Immunol., 159:4745-4752 (1997) based on the finding that antibodyresponses induced by TNP-LPS and BCL₁ tumor cells were inhibited byneutralization of IL-12. Since IL-12 is a known activator of NK cells,the role of these cells in IL-12 mediated enhancement of TI antibodyresponses was investigated. For this purpose mice that are transgenicfor the human CD3ε gene and which lack T and NK cells were used. It wasfound that exposure of these animals to a TI antigen in the presence ofIL-12 resulted in enhancement of IgG2a and IgG3 antibody responses.Thus, as described herein, NK cells are not required for stimulation ofTI IgG production by IL-12. However, NK cells do appear to be importantin maintaining IL-12-induced IgG expression over an extended period oftime. While WT and CD3ε mice showed no differences in responsiveness toIL-12 on day 14 after immunization, CD3ε mice did demonstrate lowerIgG2a responses compared to WT mice on day 28 and thereafter. Therefore,although NK cells are not strictly required for IL-12's influence, theycould be critical depending upon the time of experimental observation.

[0038] IFN-γ is known to be a switch for both IgG2a and IgG3 (Buchanan,J. M., et al., Int. Immunol., 7:1519-1528 (1995); Snapper, C. M., etal., J. Exp. Med., 175:1367-1371 (1992); Metzger, D. W., et al., Eur. J.Immunol., 27:1958-1965 (1997); Snapper, C. M., et al., Science,236:944-947 (1987); Collins, J. T., et al., Int. Immunol., 5:885-891(1993)), the major isotypes induced by IL-12, and high levels of IFN-γmRNA were detected in the spleens of mice injected with TI antigen andIL-12. Nevertheless, in the absence of the two cells types responsiblefor IFN-γ production (T and NK cells), IL-12 still significantlyenhanced TI antibody responses. This suggests that IFN-γ either is notinvolved in IL-12-mediated enhancement of TI antibody responses or isbeing produced by another cell type. B cells have been reported toproduce IFN-γ, particularly after stimulation with IL-12 and IL-18(Pang, Y. Y., et al., Blood, 80:724-732 (1992); Buschle, M. D., et al.,J. Exp. Med., 177:213-218 (1993); Yoshimoto, T., et al., Proc. Natl.Acad. Sci., USA, 94:3948-3953 (1997)). Furthermore, IFN-γ mRNA has beendetected in CD3ε spleen cells that have been activated in vitro with LPSand IL-12. To directly assess the role of IFN-γ in mediating IL-12enhancement, TI responses in IFN-γ knockout (GKO) mice were examined andit was found that enhancement of IgG2a and IgG3 by IL-12 still occurredin these mice. In fact, IgG3 secretion in response to TI antigenimmunization appeared to be totally independent of IFN-γ. In earlierstudies using TD antigens (Metzger, D. W., et al., Eur. J. Immunol.,27:1958-1965 (1997) it was similarly found that IL-12 could enhance IgGproduction in mice genetically deficient in IFN-γ expression. Productionof antibody in response to TD antigens was low in GKO mice, butinjection of IL-12 significantly enhanced IgG1 and IgG2b levels. Infact, IgG1 levels in some cases were reconstituted by IL-12 to the samelevels seen in WT mice. The mechanisms involved in IL-12 enhancement inthe absence of IFN-γ are unknown but could involve other intermediarycytokines or a direct stimulation of B cells. It has recently been shownthat IL-12 binds to the surface of activated human and murine B cells(Vogel, L. A., et al., Int. Immunol., 8:1955-1962 (1996), which suggeststhat post-switched cells can respond directly to IL-12, a mechanism thatwould be consistent with results in both TD and TI antigen systems.

[0039] The findings reported herein are significant since S. pneumoniaeand N. meningitidis are the leading causes of pneumonia, meningitis andotitis media, causing an estimated 7.5 million cases per year in theU.S. and over 100 million per year worldwide. In addition, the currentlyavailable polysaccharide vaccines and conjugate vaccines underdevelopment are of limited value particularly in the ability tostimulate isotype switching. The fact that IL-12 induces IgG2aantibodies in response to vaccination is particularly interesting sincethis is the primary isotype which mediates optimal complement fixationand opsonization in mice. The results described herein were obtainedusing complete Freund's adjuvant (CFA) and alum, the adjuvant approvedfor human use, as adjuvant. Furthermore, preliminary analyses ofantibody specificities to individual serotypes within the vaccinepreparations indicate that high levels of IgG2a are induced againstserotypes associated with the most problematic organisms. Robbins et al.(Robbins, J. B., et al., J. Infect. Dis., 171:1387-1398 (1995); Robbins,J. B., et al., Adv. Exp. Med. Biol., 397:169-182 (1996)) have providedevidence that protection against encapsulated bacteria is associatedwith levels of circulating IgG antibodies, suggesting that serum IgG2aantibodies induced by IL-12 will be effective in mediating bacterialclearance. Thus, the results described herein indicate that IL-12 isuseful for increasing the protective capacity of current polysaccharidevaccines as well as conjugate vaccines as they become available.

[0040] Thus, the methods and compositions described herein can be usedto treat and/or prevent a disease or condition associated with apathogen having one or more TI antigens. The methods and compositionsdescribed herein can utilize an effective amount of IL-12 in combinationwith a single TI antigen or multiple TI antigens which can be derivedfrom the same pathogen, from different strains of a pathogen or fromdifferent pathogens. Thus, the composition comprising IL-12 and one ormore TI antigens can be used to prevent and/or treat one or more diseaseor condition associated with the pathogen(s) from which the TIantigen(s) is derived.

[0041] The present invention is illustrated by the following examples,which are not intended to be limiting in any way.

Exemplification

[0042] Materials and Methods

[0043] Mice

[0044] Six- to eight-week old BALB/c and C57BL/6 mice were obtained fromthe National Cancer Institute (Bethesda, Md.). C57BL/6 TCR β δ doubleknockout (KO) mice, CD3ε transgenic mice, (C57BL/6J×CBA/J)F₁ mice, andBALB/c IFN-γ KO (GKO) mice were all obtained from Jackson Laboratories(Bar Harbor, Me.). The mice were housed in the animal facility at theMedical College of Ohio and all experimental procedures performed onthem adhered to an approved IACUC protocol.

[0045] IL-12 Treatment and Immunization Strategy

[0046] Recombinant murine IL-12 was provided by Genetics Institute,Cambridge, Mass. IL-12 was stored in aliquots at −80° C. until use.Groups of 3-4 mice were injected intraperitoneally (i.p.) for threeconsecutive days (days −1, 0, 1) with either 1 μg IL-12 diluted in PBScontaining 1% normal mouse serum (PBS-1% NMS) or, as a control, PBS-1%NMS vehicle only. The amounts IL-12 used did not result in any apparenttoxicity.

[0047] Mice were immunized i.p. on day 0 with antigen precipitated inalum or emulsified in complete Freund's adjuvant (CFA; Gibco BRL, GrandIsland, N.Y.) as specified in the Results. Preparation of antigen inalum was performed by mixing 300 μPBS containing 500 μg of antigen with160 μl of 10% aluminum potassium sulfate (Fisher Scientific, Pittsburgh,Pa.), adjusting the pH to 6.5, and washing the precipitate three timeswith PBS. Antigens included 50 μg/mouse of DNP-OVA and DNP-Ficoll (bothfrom Biosearch Technologies Inc., San Rafael, Calif.) as modelT-dependent (TD) and T-independent (TI) antigens, respectively. Inaddition, the following commercial polysaccharide vaccines were used: 1)115 μg/mouse of PNU-Inmmune 23 (Lederle Laboratories Division, AmericanCyanamid Company, Pearl River, N.Y.), a polyvalent pneumococcal vaccineconsisting of a mixture of purified capsular polysaccharides from 23serotypes of Streptococcus pneumoniae; and 2) 20 μg/mouse ofMenomune-A/C/Y/W-135 (Connaught Laboratories Inc., Swiftwater Pa.), ameningococcal vaccine consisting of purified capsular polysaccharidesfrom 4 serogroups of Neisseria meningitidis. In some experiments, micewere boosted i.p. with 115 μg PNU-immune 23 emulsified in incompleteFreund's adjuvant (IFA, Gibco BRL, Grand Island, N.Y.) on day 28. Serawere prepared by bleeding from the orbital plexus.

[0048] Detection of Antibody Levels by Elisa

[0049] Anti-DNP antibody levels were measured by isotype-specific ELISAsas previously described (Buchanan, J. M., et al., Int. Immunol.,7:1519-1528 (1995); Metzger, D. W., et al., Eur. J. Immunol.,27:1958-1965 (1997)) with some modifications. Briefly, microtiter plates(Nalge Nunc International, Naperville, Ill.) were coated overnight with10 μg/ml DNP-bovine serum albumin conjugate (DNP-BSA; BiosearchTechnologies, Inc.) in PBS. After washing the plates with PBS containing0.3% Brij-35 (Sigma, St. Louis, Mo.), the plates were blocked with PBScontaining 5% fetal calf serum (Hyclone Laboratories, Logan, Utah) and0.1% Brij-35 (Sigma) for 1 hour at room temperature. The plates werethen incubated with serial dilutions of mouse sera for 2 hours at roomtemperature and bound antibody was detected with alkaline phosphatasewhich was conjugated to goat anti-mouse Ig (Sigma) for detection oftotal antibody or to specific goat anti-isotype antibody (SouthernBiotechnology Associates, Birmingham, Ala.) for detection of individualisotypes. After incubation at room temperature for 1 hour, p-nitrophenylsubstrate was added and color development was read at 405 nm with anELISA microplate reader (Bio-Tek Instruments Winooski, Vt.). The isotypespecificities and appropriate working dilutions of the antibody-enzymeconjugates were determined by titration against standard myelomaproteins of known isotypes (Sigma). Specificity of the assay for DNP wasconfirmed by lack of binding of the mouse sera to BSA-coated wells.

[0050] Antibodies specific for pneumococcal and meningococcalpolysaccharides were measured by initially coating microtiter plates at37° C. for 2 hours with 100 μg/ml poly-L-lysine (PLL, Sigma) in PBS.Plates were washed with PBS and 10 μg/ml Pnu-Immune 23 or MenomuneA/C/Y/W-135 in PBS was added to each well overnight. The remainder ofthe assay was performed as described above for anti-DNP antibodymeasurement. No binding of antisera was observed using plates coatedonly with PLL.

[0051] Statistical Analyses

[0052] Statistical analyses were performed using the Mann-Whitney UTest. Titers were calculated by fitting the data to a generalized fourparameter logistics curve using Titercal Software.

[0053] Cytokine Reverse Transcriptase-PCR (RT-PCR)

[0054] Total RNA was isolated from spleens using Trizol reagent (LifeTechnologies, Inc., Gaithersburg, Mass.). cDNA synthesis was performedusing a reverse transcriptase kit (Life Technologies) utilizing oligo(dT)₁₆₋₁₈ primers. The cDNA was amplified using specific primers forIFN-γ, and hypoxanthine phosphoribosyl transferase (HPRT). The sense andantisense primers had the following sequences: IFN-γ,5′-TGAACGCTACACACTGCATCTTGG-3′ (SEQ ID NO: 1) and5′-CGACTCCTTTTCCGCTTCCTGAG-3′ (SEQ ID NO: 2); HPRT,5′-GTTGGATACAGGCCAGACTTTGTTG-3′ (SEQ ID NO: 3) and5′-GATTCAACTTGCGCTCATCTTAGGC-3′ (SEQ ID NO: 4). PCR amplification wasperformed by mixing 2 μl of cDNA, 10 μl of 300 mM Tris-HCl (pH 8.5), 75mM (NH₄)₂SO₄, 2.0 mM MgCl₂, 5 μl of 2.5 mM dNTPs (InvitrogenCorporation), 0.5 μl of Taq DNA polymerase (2.5 U; GIBCO BRL), 2 μl of20 μM primer, and DEPC water to a final volume of 50 μl. The mixture wasincubated at 95° C. for 5 minutes and then subjected to the followingamplification profile: 1 minute at 95° C., 1 minute at 56° C. and 1minute at 72° C. for a duration of 35 cycles. This was followed by afinal extension for 10 minutes at 72° C. The PCR products were separatedon a 2.5% agarose gel and stained with ethidium bromide. The bands werevisualized and photographed under UV transillumination.

[0055] Results

[0056] IL-12 Enhances DNP-Specific IgG2a and IgG3 Levels AfterImmunization with DNP-OVA or DNP-Ficoll

[0057] IL-12 has been shown to stimulate cell-mediated immunity throughincreased IFN-γ secretion by T cells and NK cells (Trinchieri, G., Annu.Rev. Immunol., 13:251-276 (1995); Buchanan, J. M. et al., Int. J.Pediat. Hematol. Oncol., 3:123-131 (1996)). However, in that study theeffects of IL-12 on humoral immunity was unclear. The ability of IL-12to significantly enhance the humoral immune response to T-dependentantigens, such as proteins and hapten carrier conjugates has beenpreviously demonstrated (Buchanan, J. M. et al., Intl. Immunol.,7:1519-1528 (1995)). The ability of IL-12 to also enhance antibodyresponses to T-independent (TI) antigens is now described herein. Asdescribed below, the influence of IL-12 on IgG antibody responses to theTI antigen, DNP-Ficoll, was observed and compared to the effects seenwith the TD form of DNP conjugated to OVA.

[0058] BALB/c mice were injected with 1 μg of IL-12 or PBS vehicle i.p.on days −1, 0, +1 and with DNP-OVA or DNP-Ficoll emulsified in CFA onday 0. It was found that IL-12 treatment of mice during immunizationwith DNP-OVA caused increased production of serum antibody within 7 daysafter immunization compared to mice receiving antigen and PBS vehicle.The observed enhancement persisted until at least day 35. Mice immunizedwith DNP-Ficoll and treated with IL-12 also showed increases in antibodylevels, although this effect was not evident until day 21 afterimmunization. Analysis of individual antibody isotypes revealedincreases in the levels of DNP-specific IgG2a and IgG3 in IL-12 treatedmice compared to control mice (FIGS. 1A-1D). Significant increases wereobserved in mice immunized with either the TD or TI forms of DNP,although the effects were most dramatic in the former group. In bothcases, the enhancement of IgG2a and IgG3 antibodies reached a maximum onday 21 and remained elevated for at least 3 additional weeks. Theresults demonstrate that the influence of IL-12 is similar in both TDand TI humoral immune responses and show that IL-12 is an effectiveadjuvant for TI polysaccharide vaccines.

[0059] IL-12 Enhances the Humoral Response of Mice to MeningococcalPolysaccharide Vaccine

[0060] The next series of experiments were performed to determine howIL-12 stimulates IgG antibody responses to other TI antigens, especiallypolysaccharide antigens that are of medical importance in humans.Experiments were performed using a meningococcal polysaccharide vaccine,a vaccine with limited efficacy in infants. For this purpose, BALB/cmice were immunized with a meningococcal polysaccharide vaccine(Menomune) consisting of the A, C, Y and W-135 capsular serogroups.Vaccine was administered i.p. to adult BALB/c mice together with 3 dailydoses of 1 μg IL-12 or PBS vehicle. The mice were bled weekly and testedfor polysaccharide-specific antibody of defined isotype by ELISA. It wasfound that levels of IgG2a and IgG3 anti-polysaccharide antibodies weredramatically enhanced by IL-12 administration compared to mice notexposed to IL-12 (FIGS. 2A-2F). In fact, the mice mounted only very weakor no IgG2a responses unless they had been inoculated with both vaccineand IL-12. Levels of total and IgG1 antibodies were somewhat increasedby IL-12 exposure, IgM was slightly suppressed, and there appeared to beno detectable effect on IgG2b production.

[0061] IL-12 Enhances the Humoral Response of Mice to PneumococcalPolysaccharide Vaccine

[0062] To test the use of IL-12 as an adjuvant for TI vaccines currentlyused in humans, mice were immunized with the pneumococcal vaccine,PNU-Immune 23, a mixture consisting of purified capsular polysaccharidesfrom 23 serotypes of S. pneumoniae. On day 0, BALB/c mice were immunizedwith vaccine emulsified in CFA. On days −1, 0 and +1, the animals werealso injected i.p. with IL-12 or 1% PBS vehicle. The mice were boostedi.p. on day 28 with vaccine emulsified in IFA together with IL-12 or 1%NMS on days 27, 28, and 29. Levels of anti-pneumococcal polysaccharideantibodies were measured weekly by ELISA. It was found that mice whichwere treated with IL-12 had enhanced levels of total antibody comparedto controls. Analysis of individual isotypes showed enhanced expressionof specific IgM, IgG1, IgG2a and IgG3 (FIGS. 3A-3E). Enhancement ofIgG2a was observed as early as day 7 of the primary response, whilelevels of IgM, IgG1 and IgG3 levels were increased after day 21. IgG2blevels, on the other hand, were undetectable in both IL-12-treated andcontrol mice throughout the course of the experiment.

[0063] Enhancement of IgG2a by IL-12 Occurs in the Absence of T Cells

[0064] To assess the involvement of T cells in mediating the effects ofIL-12 on TI antibody responses, the influence of IL-12 in micespecifically lacking T cells (i.e., C57BL/6 β⁻δ⁻ TCR KO mice) wasanalyzed. C57BL/6 WT and TCR KO mice were immunized with DNP-Ficollemulsified in CFA on day 0 and injected i.p. with IL-12 or PBS vehicleon days −1, 0 and +1. Sera were collected weekly and assayed by ELISAfor DNP-specific antibodies. The results showed that the levels of WTand TCR KO responses were essentially identical and that IL-12 hadlittle effect on total antibody production in either case (FIGS. 4A-4D).There were also no differences between WT and KO mice in the ability ofIL-12 to enhance production of IgG2a DNP-specific antibodies. IgG2alevels were detectable by day 7 and reached maximum levels by day 21.Enhancement by IL-12 was also observed at day 7 and maintained throughday 28 in both strains of mice. These results confirm the TI nature ofthe response and demonstrate that the ability of IL-12 to mediate itseffects on TI antibody responses can occur in the absence of T cells. Ithas previously been shown that activated murine and human B cellsexpress a receptor for IL-12 (Vogel, L. A. et al., Int. Immunol.,8:1955-1962 (1996)) suggesting that IL-12 may directly activate B cells.Alternatively, IL-12 may stimulate natural killer cells to secretecytokines which then cause the observed effects.

[0065] IL-12 Enhances TI Antibody Responses in Mice Lacking Both T andNK Cells

[0066] There is evidence that NK cells are responsible for regulating TIantibody responses (Bondada, S. and Garg, M., “Thymus-independentantigens. in Handbook of B and T Lymphocytes., E. C. Snow, ed. AcademicPress, San Diego. 343-370 (1994); Snapper, C. M., et al., J. Immunol.,152:4884-4892 (1994); Snapper, C. M., and Mond, J. J., J. Immunol.,157:2229-2233 (1996); Wilder, J. A., et al., J. Immunol., 156:146-152(1996); Koh, C. Y. and Yuan, D., J. Immunol., 159:4745-4752 (1997)). Itis also known that IL-12 activates NK cells (Trinchieri, G., Annu. Rev.Immunol, 13:251-276 (1995); Trinchieri, G., Blood, 84:4008-4027 (1994)).Therefore, to investigate the role of NK cells in the enhancement ofIgG2a anti-DNP responses by IL-12, mice which lack both T and NK cellswere inoculated with DNP-Ficoll and IL-12. The animals used for thisexperiment were (C57BL/6×CBA)F ₁ mice that are transgenic for the humanCD3ε gene (Jackson Labs). Introduction of this transgene has led to acomplete blockage of both T lymphocyte and NK cell development in therecipient mice but B cell development is normal (Wang, B., et al., Proc.Natl. Acad. Sci., USA, 91:9402-9406 (1994)). Unexpectedly, it was foundthat exposure of these animals to DNP-Ficoll in the presence of IL-12resulted in typical enhancement of IgG2a anti-DNP antibody responses(FIGS. 5A-5D). The level of enhancement was actually more striking inCD3ε mice compared to WT controls because of the nearly complete absenceof IgG2a antibody produced by CD3ε mice not inoculated with IL-12.Although in this experiment IL-12 showed little enhancement of IgG3anti-DNP levels in WT mice, it clearly stimulated IgG3 production inCD3ε mice (FIGS. 5A-5D). With regard to other isotypes, IL-12 treatmentof WT mice caused reduced production of IgG1 and IgG2b anti-DNP antibodyand had no effect on IgM antibody. In CD3ε mice, on the other hand,IL-12 caused suppression of IgM but had no effect on IgG1 and IgG2blevels. Taken together, the results provide evidence that the mechanismof IL-12's influence on IgG2a and IgG3 TI antibody response does notinvolve NK or T cells, although these cells might influence expressionof other isotypes. It has been previously shown that activated B cellsexpress a receptor for IL-12 (Vogel, L. A., et al., Int. Immunol.,8:1955-1962 (1996)), suggesting that IL-12 directly activates B cells.Alternatively, IL-12 could stimulate secretion of intermediary cytokinesfrom cells other than T or NK cells and these cytokines may then mediatethe observed effects.

[0067] Enhancement of TI Antiboby Production by IL-12 is only PartiallyDependent on IFN-γ

[0068] IFN-γ induced by IL-12 plays a pivotal role in enhancement ofIgG2a and IgG3 during TD immune responses (Germann, T, et al., Eur. J.Immunol., 25:823-829 (1995); Buchanan, J. M., et al., Int. Immunol.,7:1519-1528 (1995); Metzger, D., et al., Eur. J. Immunol., 27:1958-1965(1997)). To investigate the role of IFN-γ in stimulating IgG2a and IgG3antibody production during TI responses, BALB/c mice were immunized withDNP-Ficoll and injected with either PBS vehicle or IL-12 as describedabove. Analysis of splenic mRNA 12 hours later revealed that IFN-γlevels were substantially increased after exposure to IL-12. The resultswere identical regardless of whether alum or CFA was used as anadjuvant. The ability of IL-12 to induce large amounts of IFN-γ mRNAduring a TI response suggests that IFN-γ is important in the observedenhancement of antibody production.

[0069] To directly elucidate the importance of IFN-γ, WT and GKO micewere immunized with Menomune or DNP-Ficoll and simultaneously injectedwith either PBS vehicle or IL-12. WT mice treated with antigen and IL-12has a three- to ten-fold enhancement of serum IgG2a levels in comparisonto mice that received only antigen and PBS vehicle (the Table). GKO miceimmunized in the same manner showed less enhancement but still tended tohave increases in levels of IgG2a (approximately two-fold increases). Inthe case of IgG3, two to three-fold enhancement by IL-12 was observed inboth WT and GKO mice, except that GKO mice immunized with DNP-Ficollproduced large amounts of IgG3 antibody regardless of whether they weretreated with IL-12 or PBS vehicle. These results suggest thatenhancement of IgG2a by IL-12 is partially but not completely dependenton IFN-γ whereas the increase in levels of IgG3 is whollyIFN-γ-independent. Enhancement of TI Antibody Responses by IL-12 is OnlyPartially IFN-γ-Dependent Anti- body In vivo Isotype treatment WT meantiter* GKO mean titer IgG2a Menomune + 25 23 PBS (0, 18, 22, 59) (0, 29,31, 33) IL-12 243⁺ 48 (70, 112, 266, 525) (20, 43, 47, 82) DNP-Ficoll +61 57 PBS (48, 60, 65, 69) (43, 47, 55, 83) IL-12 193⁺ 111 (101, 123,147, 402) (52, 76, 113, 201) IgG3  Menomune + 250 197 PBS (110, 179,260, 452) (102, 165, 203, 317) IL-12 626⁺ 498⁺ (316, 625, 650, 911)(310, 482, 558, 643) DNP Ficoll + 473 794 PBS (328, 430, 497, 637) (342,443, 474, 1915) LL-12 740 729 (323, 497, 825, 1313) (443, 544, 631,1298)

[0070] Equivalents

[0071] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1 4 1 24 DNA Artificial Sequence PCR Primer for IFN-y 1 tgaacgctacacactgcatc ttgg 24 2 23 DNA Artificial Sequence PCR Primer for IFN-y 2cgactccttt tccgcttcct gag 23 3 25 DNA Artificial Sequence PCR Primer forHPRT 3 gttggataca ggccagactt tgttg 25 4 25 DNA Artificial Sequence PCRPrimer for HPRT 4 gattcaactt gcgctcatct taggc 25

We claim:
 1. A method of inducing an immune response to a T-cellindependent antigen in a host, which comprises administering to the hostan effective amount of interleukin-12 and the T-cell independentantigen.
 2. The method of claim 1 wherein the T-cell independent antigenis selected from the group consisting of: a carbohydrate, a lipid, aglycolipid, a carrier conjugate, a lipopolysaccharide and a phage. 3.The method of claim 2 wherein the carbohydrate antigen is apolysaccharide antigen.
 4. The method of claim 3 wherein thepolysaccharide antigen is selected from the group consisting of: abacterial capsular antigen and a bacterial cell wall antigen.
 5. Themethod of claim 1 wherein the T-cell independent antigen is frombacteria selected from the group consisting of: Streptococcuspneumoniae, Neisseria meningiditis and Haemophilus influenzae.
 6. Themethod of claim 1 wherein the immune response is a humoral immuneresponse.
 7. The method of claim 6 wherein the humoral immune responseresults in an enhanced IgG2a and IgG3 antibody response.
 8. The methodof claim 1 wherein the interleukin-12 is administered as apolynucleotide under conditions in which the interleukin-12 is expressedin vivo.
 9. A method of enhancing an immune response against a T-cellindependent antigen in a host, which comprises administering to the hostan effective amount of interleukin-12 and the T-cell independentantigen.
 10. The method of claim 9 wherein the T-cell independentantigen is selected from the group consisting of: a carbohydrate, alipid, a glycolipid, a carrier conjugate, a phosphorylcholine, alipopolysaccharide and a phage.
 11. The method of claim 10 wherein thecarbohydrate antigen is a polysaccharide antigen.
 12. The method ofclaim 11 wherein the polysaccharide antigen is selected from the groupconsisting of: a bacterial capsular antigen and a bacterial cell wallantigen.
 13. The method of claim 9 wherein the T-cell independentantigen is from bacteria selected from the group consisting of:Streptococcus pneumoniae, Neisseria meningiditis and Haemophilusinfluenzae.
 14. The method of claim 9 wherein the immune response is ahumoral immune response.
 15. The method of claim 14 wherein the humoralimmune response results in an enhanced IgG2a and IgG3 antibody response.16. The method of claim 9 wherein the interleukin-12 is administered asa polynucleotide under conditions in which the interleukin-12 isexpressed in vivo.
 17. A method of inducing an immune response toStreptococcus pneumoniae in a host, which comprises administering to thehost an effective amount of interleukin-12 and a T-cell independentantigen of Streptococcus pneumoniae.
 18. The method of claim 17 whereinthe immune response is a humoral immune response.
 19. The method ofclaim 18 wherein the humoral immune response results in an enhancedIgG2a and IgG3 antibody response.
 20. The method of claim 17 wherein theinterleukin-12 is administered as a polynucleotide under conditions inwhich the interleukin-12 is expressed in vivo.
 21. A method of inducingan immune response to Neisseria meningiditis in a host, which comprisesadministering to the host an effective amount of interleukin-12 and aT-cell independent antigen of Neisseria meningiditis.
 22. The method ofclaim 21 wherein the immune response is a humoral immune response. 23.The method of claim 22 wherein the humoral immune response results in anenhanced IgG2a and IgG3 antibody response.
 24. The method of claim 21wherein the interleukin-12 is administered as a polynucleotide underconditions in which the interleukin-12 is expressed in vivo.
 25. Acomposition comprising interleukin-12 and a T-cell independent antigen.26. The composition of claim 25 wherein the T-cell independent antigenis selected from the group consisting of: a carbohydrate antigen, alipid antigen, a glycolipid antigen, a carrier conjugate antigen, aphosphorylcholine antigen, a lipopolysaccharide antigen and a phageantigen.
 27. The composition of claim 26 wherein the carbohydrateantigen is a polysaccharide antigen.
 28. The composition of claim 27wherein the polysaccharide antigen is selected from the group consistingof: a bacterial capsular antigen and a bacterial cell wall antigen. 29.The composition of claim 25 wherein the T-cell independent antigen isfrom bacteria selected from the group consisting of: Streptococcuspneumoniae, Neisseria meningiditis and Haemophilus influenzae.